For many years light microscopes have been considered a mature technology. While there have been notable attempts to extend the capabilities of the light microscope, to date such attempts have not achieved substantial gains in performance and have generally been obtained at significantly increased costs. Vibrations in microscopes have been known as a major factor contributing to the limit on resolving power. Vibrations in the microscope frame have previously been addressed by building super rigid or heavy frames, or by constructing horizontal microscopes on massive optical bench_style frames. Other attempts to improve the vibration performance have used passive or active vibration damping tables, feet or platforms.
The generation of image contrast in microscopes is an area where there has been considerable work carried out in the past. Attempts to increase the contrast of observed biological samples have resulted in many new methods such as phase contrast, interference contrast, Hoffman modulation contrast, differential interference contrast, polarized light microscopy, darkfield microscopy and fluorescent microscopy. The challenge of generating image contrast at the extreme limit of resolution yielded such techniques as high power immersion darkfield, and ultramicroscopic illumination. Phase and interference contrast techniques introduced artifacts, some of which were asymmetrical, which made the images difficult to relate to the real structure of the samples being viewed. Darkfield and fluorescent techniques presented image information in a form that is most unfamiliar to visual capabilities, much in the same way that we are unable to extract information from a photographic or electronic “negative” image.
Attempts to gain more information about cells in real time has yielded confocal microscopy which uses high power laser light sources which scan the sample area to build a final image of the sample, and newer masked confocal techniques that can build higher speed images of live samples. In general, the frame/field rate of the confocal systems is too slow for studying the high speed motion of many components in biological systems since they exhibit high speed motion.
Attempts to yield high resolution have been based on the formula for microscopic resolution first developed by Ernst Abbe, resolution limit=wavelength of light/(k×numerical aperture of the objective). Values for k ranging from 1.6 to 2 have been accepted for over 50 years but the inventor's recent work suggests the value of k can be lowered and needs to be more fully studied when applied to improved optical systems with new methods of illumination and imaging means.
As microscope systems have become more complex, more glass surfaces created more light loss due to transmission losses in the glass elements, internal reflection and stray light. The stray light contributed to poor contrast and the internal reflections and transmission losses, together with the stray light, meant that progressively more powerful light sources were needed to produce useable image brightness. These high powered sources must propagate the light at high fluxes through the sample space since most of the lossy components are between the sample and the imaging means. Modern binocular and trinocular systems with their attendant prisms, mirrors and lenses are particularly inefficient and require higher light levels.